It was recognized in the early 1960s that by generating ions in a spatially resolved region of a surface, one could obtain atomic or molecular weight maps, or images (of ion mass-to-charge (m/z)), based on the spatial distribution of analyte and mass spectrometry detection. (R. Castaing and G. Slodzian, Microanalysis by Secondary Ionic Emission, J. Microsc. 1, 395-410 (1962)). For many years, imaging mass spectrometry was largely limited to secondary ion mass spectrometry (SIMS) whereby secondary analyte ions are produced by impinging the surface with a focused beam (<1 μm) of high-energy particles (e.g., keV Cs+or Ga+) (see M. L. Pacholski and N. Winograd, Imaging with Mass Spectrometry, Chem. Rev. 99, 2977-3005 (1999)), or by using laser microprobe mass spectrometry (LMMS) in which UV photons are used to provide direct ablation and photoionization of the analyte in a spatially-resolved mode. (L. Van Vaeck, H. Struyf, W. Van Roy, and F. Adams, Organic and Inorganic Analysis with Laser Microprobe Mass Spectrometry. Part I: Instrumentation and Methodology, Mass Spectrom. Rev. 13, 189-208 (1994); L. Van Vaeck, H. Struyf, W. Van Roy, and F. Adams, Organic and Inorganic Analysis with Laser Microprobe Mass Spectrometry. Part II: Applications, Mass Spectrom. Rev. 13, 209-232 (1994)). However, both techniques are primarily limited to the analysis of atomic ions and small molecules (typically<500 amu) and ultimately provide spatial imaging resolution that directly depends on the focusing properties of the optics (i.e., ion or photon optical elements) used to define the ionizing beam. The general principle of LMMS is illustrated in FIG. 1 (prior art), which shows a molecular weight map for an organic dye patterned onto a nitrocellulose membrane. FIG. 1 depicts an imaging mass map by LDI-TOFMS of crystal violet (hexamethyl-pararosanaline, m/z=372) deposited onto nitrocellulose. in the shape of an ampersand “&.” FIG. 1A is the optical microscopy image of the deposited material. FIG. 1B is the corresponding image obtained by LDI-TOFMS where white and black circles represent mass spectra with a signal-to-noise of less than and greater than 10 at m/z 372, respectively. Each mass spectrum represents the average of 10 laser shots and the laser spot (ellipse, ca. 50×90 μm) was translated in 95 μm increments to produce the resulting 780-pixel image. The analyte was interrogated by laser desorption/ionization time-of-flight mass spectrometry (LDI-TOFMS) by rastering the sample (via micropositioners) with respect to the laser spot (nitrogen laser, 337 nm) in 95 μm increments.
In the late 1980s, the development of matrix assisted laser desorption/ionization (MALDI) provided a means to generate gas-phase ions of large intact biomolecules (ca. 102 to 106 amu) from solid samples. (M. Karas, D. Bachmann, U. Bahr, F. Hillenkamp, Matrix-Assisted Ultraviolet Laser Desorption of Non-Volatile Compounds, Int. J Mass Spectrom. Ion. Proc. 78, 53-68 (1987)). MALDI consists of incorporating analyte molecules into the crystal lattice of a UV or IR absorbing matrix, whereby matrix and analyte molecules are desorbed and ionized upon irradiation of the sample at the appropriate matrix-absorbing wavelength. Caprioli and coworkers have described imaging mass spectrometry of peptides and proteins in thin (ca. 10-20 μm) tissue sections based on MALDI-TOFMS techniques (Caprioli U.S. Pat. No. 5,808,300; incorporated by reference herein). In this method, a homogenous layer of matrix is applied to the tissue section and then a full mass spectrum is recorded at each spatial location by moving the sample relative to the MALDI laser. (R. M. Caprioli, T. B. Farmer, and J. Gile, Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI-TOFMS,” Anal. Chem. 69, 4751-4760 (1997)). By using conventional optical arrangements (i.e., an apertured primary laser beam and field lens, the final shape of the laser beam at the sample target is defined by the slit function of the aperture and exhibits a spatial resolution limited by the diffraction properties of the optics used (in practice, typically 10-20 μm for typical ultraviolet operation). This can be described by Equation 1:d=1.22×λ/NA  (1)
where d is the diffraction limited focus diameter, λ is the wavelength, and NA is the numerical aperture of the lens. (See for example, D. Malacara and Z. Malacara, “Diffraction in Optical Systems,” Chapter 9 in Handbook of Lens Design, Marcel Dekker Inc., New York (1994)).
Recent advances in MALDI optics include the application of near-field scanning optical microscopy (NSOM; See for example, E. Betzig, J. K. Trautman, T. D. Harris, J. S. Weiner, and R. L. Kostelak, Breaking the Diffraction Barrier: Optical Microscopy on a Nanometric Scale, Science 251, 1468-1470 (1991) and references therein). This techniques help to overcome diffraction-limited spatial resolution. The use of NSOM techniques for LMMS was recently demonstrated for the analysis of small organic ions, such as dihydroxybenzoic acid and acetylcholine D (see A. Kossakovski, S. D. O'Connor, M. Widmer, J. D. Baldeschwieler and J. L. Beauchamp, Spatially Resolved Chemical Analysis with an NSOM-based Laser Desorption Microprobe, Ultramicrosc. 71, 111-115 (1998)), for anthracene and bis(phenyl-N,N-diethyltriazene) ether (see R. Stöckle, P. Setz, V. Deckert, T. Lippert, A. Wokaun, and R. Zenobi, Nanoscale Atmospheric Pressure Laser Ablation-Mass Spectrometry, Anal. Chem. 73, 1399-1402 (2001)), and for peptides and oligosaccarhides by MALDI with a spatial resolution<500 nm (see B. Spengler and M. Hubert, Scanning Microprobe Matrix-Assisted Laser Desorption Ionization (SMALDI) Mass Spectrometry: Instrumentation for Sub-Micrometer Resolved LDI and MALDI Surface Analysis, J. Am. Soc. Mass Spectrom. 13, 735-748 (2002)). Note that NSOM techniques require the physical aperture of the transmitted light be placed at a distance substantially closer to the image plane (i.e., sample surface) than the wavelength of transmitted light. For example, at UV wavelengths commonly used in MALDI applications, the aperture must be placed less than ca. 350 mn from the target surface. Experimentally, this is exceedingly challenging in MALDI where the sample topography can easily exceed micrometer(s) deviation in elevation unless stringent and difficult sample preparation procedures are used. Further, NSOM techniques are currently limited to generating symmetrical (typically round) spot shapes at the image plane (i.e., sample target) and cannot be easily changed to user defined dimensions or shape.
In 1986, Hornbeck described an innovative optical element for the spatial patterning of light based on digital micro-mirror arrays (DMAs) (Hornbeck, U.S. Pat. No. 4,566,935; incorporated by reference herein). The DMA consists of highly reflective aluminum micro-mirror elements (e.g., 10-20 μm on each side) that are typically constructed in an array (e.g., 1024×768 mirrors) format. By addressing each individual mirror via a bias voltage, the relative angle of each mirror (ca. +10° to −10°, relative to normal of the array) can be positioned via a torsion hinge and rapidly switched (ca. 10-20 μs) representing an “on” or “off” state. DMA devices have found widespread application in video imaging, projection, and telecommunications, and have more recently been used in analytical spectroscopy (see D. Dudley, W. Duncan, and J. Slaughter, Emerging Digital Micromirror Device (DMD) Applications, White Paper, DLP Products New Applications, Texas Instruments, Inc. Plano, Tex. 75086). For example, Winefordener and colleagues have described using a linear DMA array (2×420 mirror array) to construct a flat-field visible wavelength spectrometer. (E. P. Wagner II, B. W. Smith, S. Madden, J. D. Winefordner, and M. Mignardi, Construction and Evaluation of a Visible Spectrometer Using Digital Micromirror Spatial Light Modulation, Appl. Spectrosc. 49, 1715-1719 (1995)). In this instrument, light dispersion and collimation is achieved by a Rowland-type curved grating spectrograph and wavelength selectivity is obtained by placing a DMA at the focus plane of the spectrograph and selectively reflecting portions the spectrum onto a photomultiplier tube detector. Later, Fateley and coworkers described the use of DMAs for constructing Hadamard transform masks for multiplexed Raman imaging (see R. A. DeVerse, R. M. Hammaker, and W. G. Fateley, Hadamard Transform Raman Imagery with a Digital Micro-Mirror Array, Vib. Spectrosc. 19, 177-186 (1999); W. G. Fateley, R. M. Hammaker, and R. A. DeVerse, Modulations Used to Transmit Information in Spectrometry and Imaging, J. Mol. Struct. 550-551, 117-122 (2000)), and multiplexed near infrared flat-field spectroscopy (see R. A. DeVerse, R. M. Hammaker, and W. G. Fateley, Realization of the Hadamard Multiplex Advantage Using a Programmable Optical Mask in a Dispersive Flat-Field Near-Infrared Spectrometer, Appl. Spectrosc. 54, 1751-1758 (2000)). By using a DMA to affect a dynamic Hadamard matrix mask, limitations of moving fixed optical matrix masks could be overcome (e.g., slow translation times, positioning errors, differences in axial position owing to stacked masks, and fixed mask element sizes). Importantly, the Hadamard transform provided enhanced signal-to-noise over conventional scanning techniques (ca. a factor of 12-14) in good agreement with that predicted from theory (see F. C. A. Dos Santos, H. F. Carvalho, R. M. Goes, and S. R. Taboga, Structure, Histochemistry, and Ultrastructure of the Epithelium and Stroma in the Gerbil (Meriones unguiculatus) Female Prostate, Tissue & Cell 35, 447-457 (2003)).